Isabelle Lamrissi-Garcia

HIF-2α Protects Human Hematopoietic Stem/Progenitors and Acute Myeloid Leukemic Cells from Apoptosis Induced by Endoplasmic Reticulum Stress

Hematopoietic stem and progenitor cells (HSPCs) are exposed to low levels of oxygen in the bone marrow niche, and hypoxia-inducible factors (HIFs) are the main regulators of cellular responses to oxygen variation. Recent studies using conditional knockout mouse models have unveiled a major role for HIF-1α in the maintenance of murine HSCs; however, the role of HIF-2α is still unclear. Here, we show that knockdown of HIF-2α, and to a much lesser extent HIF-1α, impedes the long-term repopulating ability of human CD34(+) umbilical cord blood cells. HIF-2α-deficient HSPCs display increased production of reactive oxygen species (ROS), which subsequently stimulates endoplasmic reticulum (ER) stress and triggers apoptosis by activation of the unfolded-protein-response (UPR) pathway. HIF-2α deregulation also significantly decreased engraftment ability of human acute myeloid leukemia (AML) cells. Overall, our data demonstrate a key role for HIF-2α in the maintenance of human HSPCs and in the survival of primary AML cells.

A clinical-scale expansion of mobilized CD34+ hematopoietic stem and progenitor cells by use of a new serum-free medium

BACKGROUND:  The autologous transplantation of CD34+ cells expanded ex vivo in serum‐free conditions dramatically reduces postmyeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum‐free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte–colony‐stimulating factor (G‐CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical‐grade IS medium is no longer available, a new serum‐free medium, Maco Biotech HP01 (Macopharma), was evaluated.

A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines

Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells.

Effective Gene Therapy of Mice with Congenital Erythropoietic Porphyria Is Facilitated by a Survival Advantage of Corrected Erythroid Cells

Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut248) mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.

Highly Efficient Lentiviral Gene Transfer in CD34+ and CD34+/38−/lin− Cells from Mobilized Peripheral Blood after Cytokine Prestimulation

Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34+ cells and the stem/progenitor‐cell‐enriched CD34+/38−/lin− cell subset isolated from mPB. In this study, we used an optimized third‐generation self‐inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor lα promoter. This lentivector was first used to compare multiplicity of infection (MOI)‐dependent gene transfer efficiency in cord blood (CB) versus mPB CD34+‐derived cells, colony‐forming cells (CFCs), and long‐term culture‐initiating cells (LTC‐ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans‐duction. Effects of a cytokine prestimulation period (18 hours) with interleukin‐3, stem cell factor, Flt‐3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC‐ICs derived from mPB CD34+ cells. Transduction levels in those conditions demonstrated a two‐ and fourfold increase in CFCs and LTC‐ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34+/38−/lin− cells isolated from mPB, with up to >85% of colonies derived from LTC‐ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.

Study of CCN3 (NOV) and DDR1 in normal melanocytes and vitiligo skin

Abstract:  We have hypothesised that melanocytes disappear in vitiligo because they are weakly attached to the epidermal basal membrane (melanocytorrhagy). In the epidermis, attachment of melanocytes to collagen IV is mediated through DDR1, which is under the control of CCN3. DDR1 genetic variants have been associated with vitiligo in patients of different ethnic origin. In vitro studies have shown that inhibition of CCN3 induces the detachment of melanocytes. We have studied in parallel the expression of CCN3 and DDR1 in lesional and perilesional skin of patients with vitiligo and the impact of the silencing of CCN3 and DDR1 in normal human melanocytes on their behaviour in epidermal reconstructs. Our in vivo study provides evidence of a dysregulation of the DDR1–CCN3 interaction in vitiligo skin as melanocytes remaining in perilesional skin did not express CCN3. Expression of DDR1 was decreased in lesional versus perilesional vitiligo skin in the majority of patients, and the expression of collagen IV was found decreased in all patients. Silencing of CCN3 in melanocytes induced a significant inhibition of cell adhesion to collagen IV whereas melanocytes transduced with shDDR1 still adhered well on collagen IV and did not increase melanocyte loss in epidermal reconstructs as compared with normal melanocytes. Melanocyte detachment was observed but not in all reconstructs using CCN3 silenced melanocytes. Overall, our study confirms that a downregulation of CCN3 is implicated in melanocyte adhesion in part through DDR1. In vitiligo skin, the interaction of CCN3 with other molecules, such as TGFβ and CCN2, needs to be addressed.

Modeling of congenital erythropoietic porphyria by RNA interference: a new tool for preclinical gene therapy evaluation.

Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector‐mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS‐deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients.